Proteomics can be most broadly defined as the systematic analysis and documentation of the proteins in biological samples. It is a field that has leapt to prominence within the last two years and is widely expected to have a major impact on biotechnology.
Proteomics is a mass-screening approach to molecular biology, which aims to document the overall distribution of proteins in cells, identify and characterise individual proteins of interest, and ultimately to elucidate their relationships and functional roles. Such direct, protein-level analysis has become necessary because the study of genes (by genomics) cannot adequately predict the structure or dynamics of proteins. This has been a problem for biotechnology, for it is at the protein level that most regulatory processes take place, where disease processes primarily occur and where most drug targets are to be found. There is, however, a strong and synergistic relationship between proteomics and genomics: the two disciplines investigate the molecular organisation of the cell at complementary levels (proteins and genes) and each discipline provides information that increases the effectiveness of the other.
Proteomic analysis typically involves a sequence of technologies that separate, map and then characterise proteins. The two most widely used technologies in contemporary proteomics are two-dimensional gel electrophoresis (2DGE) for protein separation and mapping, and mass spectrometry (MS) during protein characterisation. Both these stages are heavily dependent upon bioinformatics. Most of the hardware technologies used in today's proteomics are not fundamentally new, but improved design coupled with automation, integration of successive stages, miniaturisation, robotics and the use of several systems in parallel have enabled proteomics to subject proteins to high-throughput screening (HTS) on an unprecedented scale.
2DGE is currently the most powerful means of separating proteins within complex mixtures. The major breakthrough in gel design was the development of immobilised pH gradient (IPG) gels, which allow proteins to be resolved reliably to the same location of different gels at laboratories throughout the world. This has led to the standardisation of protein mapping. However, 2DGE still needs to be improved in its ability to detect hydrophobic proteins (eg transmembrane proteins), high molecular weight proteins, or low copy number proteins, many of which may be functionally important.
Protein characterisation is a dynamic and highly innovative field with the available technologies being subjected to regular adaptation. The most widely used technology for elucidation of the primary amino acid sequence of proteins is peptide mass fingerprinting, in which data generated by MS is used to interrogate protein or genomic databases. MS can also be used in the elucidation of post-translational modifications. Traditional protein analysis methods such as Edman sequencing remain in use and are themselves subject to continued innovation.
Bioinformatics, including protein and gel analysis software and the construction and interrogation of databases, is axiomatic to the entire proteomics enterprise. A number of public databases for both 2DGE maps and protein structures can be accessed on the Internet. A plethora of software services to analyse proteomics data are also freely available. In addition, an increasing number of companies are marketing proprietary databases and software solutions.
Proteomics has a wide range of applications, arising largely from the field's unparalleled ability to analyse simultaneously the response of large numbers of proteins to changes in cell state. Applications include:
It may also be possible to subtype individuals to predict responses to specific therapies. These applications have great potential within biotechnology, and particularly within the pharmaceutical industry where they are likely to have a major impact on R&D, preclinical development and clinical trials.
Proteomics faces a number of significant challenges. The current, 2DGE/MS-based paradigm is very powerful but is considered by some to be labourious and would certainly benefit from greater sensitivity and higher throughput volumes than those achieved to date. Innovations in all the key technologies, coupled with the continuing drive towards robotics and automation, are likely to address these goals effectively. In addition, however, new technologies may have a major impact on the future shape of proteomics. Alternative separation techniques to 2DGE may bypass the need for gels, and novel protein extraction techniques, for instance the use of antibody screens raised from phage libraries, may provide powerful new approaches which combine protein separation and identification. Particularly interesting is the emergence of protein chip technology, which has the potential to isolate, identify and screen the function of proteins. There is indeed likely to be a general broadening of the scope of proteomics, in which mass screening for function, in addition to protein distribution and structure, becomes more of a reality.
The proteomics sector is healthy and expanding rapidly. It is led by specialist proteomics houses, but other biotechnology companies, especially genomics and bioinformatics companies, are investing heavily in the new approach. Pharmaceutical companies with in-house biotechnology interests are following suit, and many new companies combining proteomics and genomics expertise are to be expected. Proteomics has therefore moved rapidly to establish an important niche in the biotechnology world.
CONTENTS
LIST OF TABLES
LIST OF FIGURES
GLOSSARY AND ABBREVIATIONS
EXECUTIVE SUMMARY
CHAPTER 1 INTRODUCTION
1.1 Defining proteomics
1.1.1 Genome and proteome
1.1.2 How does the proteome differ from the genome?
1.2 Rationale for protein-level analysis
1.2.1 Genetic code and protein structure
1.2.1.1 Splice variants
1.2.1.2 Post-translational modifications and intracellular processing
1.2.1.3 Functional interactions and metabolism
1.2.2 Gene activity and protein abundance
1.2.3 Genetic data versus molecular information
1.2.4 Summary
1.3 The main elements of proteomics
1.3.1 Study systems and goals of analysis
1.3.2 Analytical stages
1.3.2.1 Protein separation/mapping
1.3.2.2 Protein identification/characterisation
1.3.3 Bioinformatics and databases
1.3.4 Robotics and high-throughput screening
1.4 The world of proteomics
1.4.1 Relations with other new areas of biotechnology
CHAPTER 2 PROTEIN ANALYSIS IN PROTEOMICS
2.1 Protein separation and analysis: two-dimensional gel electrophoresis
2.1.1 Sample preparation for 2DGE
2.1.2 Approaches to 2DGE
2.1.2.1 ISO-DALT
2.1.2.2 Non-equilibrium pH gradient electrophoresis
2.1.2.3 Isoelectric focusing using immobilised pH gradients
2.1.3 Gel staining
2.1.4 Limitations of contemporary 2DGE
2.2 Protein characterisation
2.2.1 Mass spectrometry
2.2.1.1 Ionisation and detection techniques
2.2.1.2 Digestion and preparation of proteins
2.2.1.3 Peptide mass fingerprinting
2.2.1.4 Sequence tags
2.2.1.5 Finding unknown genes
2.2.1.6 Identifying post-translational modifications
2.2.1.7 Other applications of MS
2.3 Other protein separation and analysis techniques
2.3.1 Protein sequencing
2.3.2 Separation techniques
2.3.3 Antibody-based techniques
2.3.4 Protein chips
2.4 Robotics, automation and high-throughput strategies
2.4.1 Robotics and serial integration
2.4.2 Parallelism and miniaturisation
2.4.3 Challenges for high-throughput screening in proteomics
CHAPTER 3 BIOINFORMATICS IN PROTEOMICS
3.1 Gel analysis
3.1.1 Software packages
3.1.1.1 Melanie II 2-D
3.1.1.2 Phoretix-2D
3.1.1.3 Molecular Analyst
3.1.1.4 GELLAB
3.1.1.5 Flicker
3.1.2 Gel databases
3.1.2.1 Aarhus human and mouse databases
3.1.2.2 Large Scale Biology Corporation's human databases
3.1.2.3 The yeast protein database
3.1.2.4 SWISS-2DPAGE database
3.1.2.5 Tokyo Research Institute for Biological Sciences: plant databases
3.2 Protein characterisation
3.2.1 Protein characterisation websites
3.2.1.1 ExPASy molecular biology server
3.2.1.2 National Center for Biotechnology Information
3.2.1.3 EMBL Peptide and Protein Group
3.2.1.4 DBCAT
3.2.2 Protein characterisation tools and services
3.2.2.1 Entrez
3.2.2.2 PeptideSearch
3.2.2.3 ProFound
3.2.2.4 MOWSE
3.2.2.5 BLAST
3.2.3 Protein databases
3.2.3.1 SWISS-PROT and TrEMBL
3.2.3.2 OWL
3.2.3.3 Protein Data Bank
3.2.3.4 Public access genomic databases
3.3 The commercialisation of bioinformatics
CHAPTER 4 CURRENT APPLICATIONS OF PROTEOMICS
4.1 The output of proteomics
4.2 Scientific applications
4.2.1 Cell function, perturbation and pathogenesis
4.2.2 Drug modes of action
4.2.3 Toxicology
4.2.4 Markers of disease and response
4.2.5 Therapeutics
4.3 Industrial applications of proteomics
4.3.1 Proteomics and the pharmaceutical industry
4.3.2 Proteomics and the agricultural industry
CHAPTER 5 THE FUTURE OF PROTEOMICS - A SUMMARY
5.1 The shape of the industry and conceptions of proteomics
5.2 Challenges and responses
5.2.1 Gel-based proteomics
5.2.2 Protein structure and bioinformatics
5.2.3 High-throughput screening
5.3 Possible new developments
5.3.1 Functional screening
5.3.1.1 Industrialisation of the yeast two-hybrid assay
5.3.1.2 'Perturbagens'
5.3.1.3 Protein isolation/functional screening using microarrays
5.4 Output and applications
CHAPTER 6 COMPANY PROFILES
6.1 Specialist proteomics companies
6.1.1 Oxford GlycoSciences
6.1.2 Large Scale Biology Corporation
6.1.3 Ciphergen Biosystems
6.1.4 Biovation Ltd
6.1.5 Proteomix
6.1.6 Proteome Inc
6.1.7 Genome Pharmaceuticals Corporation
6.2 Bioinformatics companies with strong interests in proteomics
6.2.1 Incyte
6.2.2 Phoretix International
6.2.3 Oxford Molecular Group
6.2.4 Structural Bioinformatics
6.2.5 GeneBio
6.2.6 GeneData
6.2.7 Pangea Systems
6.3 Biotechnology companies with strong interests in proteomics
6.3.1 MitoKor
6.3.2 Pharmagene Laboratories Ltd
6.3.3 Ventana Genetics
6.3.4 Millennium Pharmaceuticals
6.3.5 Amgen
6.3.6 Genetics Institute
6.3.7 Human Genome Sciences
6.3.8 Cambridge Antibody Technology
6.4 Providers of services and equipment for proteomic analysis
6.4.1 ProteiGene
6.4.2 Genomic Solutions
6.4.3 Protana
6.4.4 Radius Biosciences
6.4.5 PerSeptive Biosystems/Perkin-Elmer
6.4.6 ESA
6.4.7 Bio-Rad Laboratories
6.4.8 Amersham Pharmacia Biotech
6.5 Major pharmaceutical companies with in-house proteomics programmes
REFERENCES
APPENDIX 1 PROTEOMICS AND THE INTERNET
A.1 General proteomics/biotechnology sites
A.2 Links to websites for gels and 2DGE
A.3 Gel analysis tools
A.4 Gel databases
A.5 Protein analysis tools and facilities
A.6 Protein databases
A.7 Public access genomic databases
A.8 Selected proteomics companies
A.9 Other useful sites
LIST OF TABLES
Table 4.1 Potential roles of proteomics in drug development
LIST OF FIGURES
Figure 1.1 Non-linearity of relations between the genome and proteome
Figure 1.2 Typical analytical stages in proteomics
Figure 1.3 Relationship of proteomics to other new biotechnology disciplines
Figure 2.1 MALDI-TOF mass spectrometry
Figure 3.1 Page from the Aarhus human keratinocyte gel database
Figure 3.2 Web interface for a typical internet-based protein structure research tool,
MOWSE
Figure 4.1 General scientific applications of proteomics
Published: December 1998
Ref: BS982E
Pages: 60+
Price: £143/$300/¥35,000
© PJB Publications Ltd. 2001 All rights reserved. |